A known method of obtaining yeast strains harbouring multiple copies of a desired gene is in the use of multicopy plasmids. Plasmids, however have several disadvantages: (i) the expression level expressed in terms of the ratio of desired protein produced to copy number of the desired gene is generally lower for plasmids than for integrated copies of the desired gene; thus, plasmids impose a bigger metabolic burden to the cell than integrated copies, which may result in a penalty in terms of biomass accumulation; (ii) plasmids are prone to structural instability which may lead to loss of the desired gene from the plasmid or to a partial protein product; (iii) plasmids may give rise to segregational instability leading to plasmid loss. The latter especially occurs if high level expression is desired which imposes a strong metabolic burden to the cell.
To overcome mitotic instability associated with plasmids, integration of multiple copies of a desired gene has met with some success in yeast. Multiple copies of desired genes have been integrated at a single integration site in the genome, mostly in the ribosomal DNA of the yeast genome. Multicopy integration at a single locus is almost invariably achieved by the integration of multiple copies in tandem in the yeast genome; this easily leads to instability due to out-recombination of copies of the construct as a consequence of the presence of direct repeats flanking the construct containing the desired gene. The more copies that are found in tandem, the easier it leads to excision by out-recombination of single copies, thereby decreasing protein production levels to an unpredictable, but lower, level.
To overcome some of the instability, a system was devised to obtain integration of high copy numbers in the ribosomal DNA by using a deficient selection marker, for example wherein a weak promoter is cloned in front of the selection marker (EP 0 481 008). A first disadvantage is the need of selection markers in the production strain. Dominant antibiotic-resistance markers are less desirable from a regulatory standpoint whereas on the other hand auxotrophic markers are often not useful because they require mutant recipient host strains. Moreover, the presence of selection markers may also add to an unwanted complexity of the fermentation. Secondly, the ribosomal RNA encoding DNA locus is found in a nuclear organelle called the nucleolus, which is not an ideal location for obtaining optimal expression of a protein-encoding gene, as protein encoding genes are RNA-polymerase-II transcribed genes.
It is an object of the invention to provide for yeast cells having multiple copies of a desired gene integrated into the genome without some or all of the problems encountered with yeast cells known in the prior art.